DNA methylation biomarkers in asthma and rhinitis: Are we there yet?

Abstract The study of epigenetics has improved our understanding of mechanisms underpinning gene‐environment interactions and is providing new insights in the pathophysiology of respiratory allergic diseases. We reviewed the literature on DNA methylation patterns across different tissues in asthma and/or rhinitis and attempted to elucidate differentially methylated loci that could be used to characterize asthma or rhinitis. Although nasal and bronchial epithelia are similar in their histological structure and cellular composition, genetic and epigenetic regulation may differ across tissues. Advanced methods have enabled comprehensive, high‐throughput methylation profiling of different tissues (bronchial or nasal epithelial cells, whole blood or isolated mononuclear cells), in subjects with respiratory conditions, aiming to elucidate gene regulation mechanisms and identify new biomarkers. Several genes and CpGs have been suggested as asthma biomarkers, though research on allergic rhinitis is still lacking. The most common differentially methylated loci presented in both blood and nasal samples are ACOT7, EPX, KCNH2, SIGLEC8, TNIK, FOXP1, ATPAF2, ZNF862, ADORA3, ARID3A, IL5RA, METRNL and ZFPM1. Overall, there is substantial variation among studies, (i.e. sample sizes, age groups and disease phenotype). Greater variability of analysis method detailed phenotypic characterization and age stratification should be taken into account in future studies.


| INTRODUCTION
Respiratory diseases associated with allergy, such as asthma and rhinitis, constitute a major and continuously growing public health concern specially in the western world. Notably, all atopies combined, now affect approximately 20% of the global population. 1 Asthma is unanimously accepted as an important socioeconomic issue that affects roughly 300 million people. Allergic rhinitis (AR) shows an everincreasing prevalence on a global scale with more than 600 million patients. 2 A considerable percentage present severe morbidity which medication fails to control, leading in a sharp decrease of their life quality. These statistics are bound to increase and rhinitis along with asthma appear as major epidemics of current time and rapid initiatives need to be taken towards the direction of prevention, therapeutics and disease management. Evidence indicates that etiology of asthma and allergic diseases is complex and has strong genetic and environmental components. Since the epigenome is modified by environmental factors, epigenomic and transcriptomic profiling may provide added value for individual prediction models of asthma outcomes, in addition to genomic profiling. 3 Epigenetic mechanisms provide a new understanding of geneenvironment interactions. Modifications to the epigenome mediate endogenous or exogenous environmental exposures on immune development. 4 In mammals, DNA methylation regulates gene expression and ensures genome stability. Methylation almost exclusively occurs in the context of the CpG dinucleotide. In promoters and other cis-regulatory sequences (i.e. enhancers, insulators) DNA methylation may hamper transcription factor binding, further contributing to gene silencing. 5 During fetal life and after birth, DNA methylation continues to play a pivotal role in cellular commitment and differentiation. [6][7][8] Besides these programmed changes, gain and loss of DNA methylation occur in various genomic regions as a consequence of cellular and environmental stresses and stochastic changes during lifetime. Because the respiratory system is commonly exposed to environmental stimuli (chemicals, dust, bacteria, viruses, etc.), the epigenome of the airway cells is prone to dynamic changes that may, ultimately, affect gene expression. DNA methylation can be assessed either over the entire genome (whole-genome methylation profiling) or by candidate studies designed to search specifically for differentially methylated regions (DMRs) or CpGs in specific genes or regions in the DNA. 3

| AGE-DEPENDENT EPIGENETIC CHANGES
One of the most prominent external factors influencing DNA methylation changes is aging and it has been reported that the chronological age can be determined by DNA methylation patterns. 9 Aberrant DNAm level of aging-related genes has been recorded in asthma patients. 10 Accelerated epigenetic aging, meaning the difference between epigenetic age and chronological age, has been associated with a large number of disease and an overall greater risk of death 9 while longevity has been associated with decelerated epigenetic aging. 11 The development of allergic diseases is likely agedependent during childhood 12 ; however, chronological age alone does not fully explain disease variability. Epigenetic aging has now also been assessed in the context of atopic or allergen sensitization and asthma using a variety of different clocks. 13,14 There are several methods available to estimate epigenetic aging, 9,[15][16][17][18] and among them, the Horvath method for epigenetic age estimation (DNAmAge) is used widely and has shown high accuracy. 19 Data have shown a significant epigenetic age acceleration in children with current asthma (0.74 years) and even greater age acceleration for children with allergic asthma (1.30 years). For every 10-fold increase in FeNO, the epigenetic age was accelerated by 1.11 years. In total, epigenetic age of nasal samples is accelerated by asthma and is correlated with elevated biomarkers of allergic disease such as IgE and FeNO. 13 Furthermore, accelerated epigenetic aging in children at 7-8 years of age was associated with increased serum IgE levels and a 1.2-to 1.3fold increased risk of atopic sensitization, or sensitization to environmental or food allergens for every year increase in epigenetic age. 14 Machine learning approaches are increasingly used to address healthcare problems; up to date, only one study has been conducted to predict lung functions using machine learning approaches by utilizing the effect of DNAmAge and accelerating age on lung function.
Arefeen et al. 20 suggested that apart from the previously described factors height, weight, and sex, changes in epigenetic age acceleration between 10 and 18 years can improve the prediction of FEV1 and FVC at 18 years of age and proposed five selected regression models for machine learning techniques to be used for lung function prediction.
DNA methylation patterns are tissue specific, and one critical limitation for human epigenetic studies is that tissues that are relevant for disease etiology cannot be easily obtained from patients and study participants. 21 Various biological specimens have been used to analyze DNA methylation in airway diseases such as sputum, bronchoalveolar lavage (BAL) and blood samples. Overall, nasal and bronchial pseudostratified epithelia are similar in their histological structure and cellular composition 5 ; however, genetic and epigenetic regulation may differ across tissues.
A literature review was conducted in PubMed database for articles published up to August 2021. The search terms used were 'asthma' or 'allergic asthma' or 'allergic rhinitis' or 'allergic respiratory diseases' and 'epigenetics' or 'DNA methylation' or 'epigenome wide association study'. The identified studies were divided into categories regarding the type of tissue used, that is, bronchial epithelial cells, nasal epithelial cells and blood cells. This review aims to describe DNA methylation patterns across different tissues which are associated with allergic respiratory diseases such as asthma and rhinitis. Using a state-of-the-art perspective, including the concept of epigenetic aging and machine learning, we try to elucidate differentially methylated loci that could be used as immune age biomarkers.

| DIFFERENTIAL DNA METHYLATION IN BRONCHIAL EPITHELIAL CELLS
Over the last decade, 11 relevant studies were identified, which assessed the methylation status of the genome in asthmatic bronchial epithelial cells (BECs), the primary cell type exposed to inhalants, and the corresponding effect on gene expression (Table 1). BECs are the primary cell type exposed to inhalants, but their location makes collection more technically challenging compared to nasal cells, hence less studies are inclined to include them. Nevertheless, several CpGs were identified as having an altered methylation status, along with various DMRs, both when comparing asthmatics to controls, as well as different asthma subgroups to each other. Although not always, this difference in methylation was often found to directly or indirectly affect proximal or distal gene expression levels. Various sample sizes were employed, however, most had n ≤ 25, and only three were larger with n > 50. Apart from two studies that included children, most focused on adults, roughly between their twenties and forties, and used the Illumina 450k Beadchip, on par with the rest of the epigenome field.
Notably, two of the three largest studies thus far, were conducted by the same research group. 24,26 The first looked into the epigenetic response of cultured BECs to interleukin 13 (IL-13), a key cytokine involved in asthma pathogenesis, 31 and identified in the IL-13-treated cells an epigenetic fingerprint consisting of 6522 differentially methylated CpG sites (44% hypermethylated, 56% hypomethylated) compared to controls, most of which (77%) were near a gene body (41%), or gene transcription start site (36%), totaling 3771 genes. Some genes were associated with multiple of these CpGs, with the authors singling out Tenascin B (TNXB), a member of the tenascin family of extracellular matrix glycoproteins with anti-adherence effects, due to the presence of 12 hypomethylated CpGs, as well as Chitinase 3-like 1 (CHI3L1), an asthma biomarker. 32 Gene expression analysis revealed extensive transcriptomic changes, with 63% of assessed genes (8524/13,532) being differentially expressed (52% increase, 48% decrease), with the authors singling out Chemokine (C-C motif) ligand 26 (CCL26), a chemokine elevated in asthmatic airways, as well as the T helper 2-high asthma biomarkers Periostin (POSTN) and Serpin Family B Member 2 (SERPINB2). 33,34 Interestingly, 21% of genes within 1500 kb of an assayed CpG site were in or near a minimum of one IL-13-responsive CpG site, and were significantly enriched for genes associated with asthma. When the methylation status of the 6522 previously identified IL-13-responsive CpG sites were subsequently assessed in freshly isolated cells from asthmatics, 31% were found to also be differentially methylated compared to controls, 74% of which had the same direction of methylation effect compared to the cell culture model. Lastly, a weighted gene co-expression network analysis identified two clusters of highly correlated genes which correlated with clinical phenotypes of either asthma severity and lung function or eosinophilia. These results suggest that part of the epigenetic variation seen in asthmatics may be induced by IL-13, via persistent methylation changes in asthmatic airways.
The same group subsequently published a study 26 which presented a full analysis of the comparison between asthmatics and controls from mostly the same population which they previously used to compare the results with their IL-13 in vitro model. They identified 40,892 differentially methylated CpG-sites in asthmatics (54% hypermethylated, 46% hypomethylated), and gene expression analysis showed the DMRs modestly correlated with their nearest gene expression, including asthma-associated genes, with the authors singling out the previously mentioned CCL26, and Mucin 5AC (MUC5AC), with roles in airway defense against particulates/pathogens. Furthermore, a linear model framework showed 9.89% among all CpGs within 5kb of a SNP and 11.96% of DMRs were associated with at least one methylation quantitative trait locus.
Out of the remaining studies comparing asthmatics to controls, one study found no difference in methylation levels, 22 and another measured similar overall methylation levels, with the only difference being a CpG motif in the promoter of Lipocalin 6 (LCN6), which is involved in male fertility. The rest found significant differences, with a study identifying 864 DMRs associated with 428 genes, 28  (CREBBP) and E1A Binding Protein P300 (EP300). Notably, regarding genes involved in epigenetic processes, Tet Methylcytosine Dioxygenase 1 (TET1), which is involved in DNA demethylation, was found to be methylated in another study which used in air-liquid interface cultures of asthmatic BECs, 30 whereas Protein Arginine Methyltransferase 1 (PRMT1), a histone methyltransferase, was not. The same study also found higher global methylation levels in asthmatic BECs, and their list of the top 100 group of highly methylated genes includes the previously mentioned LCN6, CCL26, CREBBP and TNXB, with several of the rest of the genes associated with cytoskeletal remodeling, cell growth, ion transport, metabolism, T-cell signaling pathway, and bronchial barrier regulation (Table 1).
When comparing atopic to non-atopic asthmatics, one study found no difference between the methylation signatures, 22 however when it compared non-asthmatic atopic children to asthmatic it did find 8 differentially methylated CpG sites associated with 8 genes: Early Growth Response 4 (EGR4), S100 Calcium Binding Protein A2   promoter hypomethylation, decrease in ZPBP2 promoter methylation from 16% to 5%, decrease in GSDMA promoter methylation from 57% to 25%, with GSDMA-CG1 promoter methylation reduced from 9% to 0% (previously found to be hypomethylated in asthmatic T A B L E 2 (Continued)  35 The study also demonstrated how changes in DNA methylation could affect allele expression ratios, specifically finding changes in allelic bias of ZPBP2 and ORMDL3.
Overall, although the findings between the studies were not highly comparable, a somewhat common finding was the differential DNA methylation of epithelial barrier genes responsible for functions including adhesion and immune response regulation, as well as genes responsible for cell proliferation, migration and differentiation, along with several epigenetic modifier genes all of which is relevant in asthma, since the condition is characterized by airway hyperresponsiveness, airway wall remodeling and airway inflammation.

| DNA METHYLATION IN NASAL EPITHELIAL CELLS (NECS)
Cells of the nasal epithelium have properties resembling bronchial epithelial cells and nasal brushing is much less invasive than bronchial brushing or BAL; thus, this technique represents a good surrogate model for lower respiratory tract studies. 36,37 There is evidence that the study of the nasal methylome allows for making reliable conclusions about DNA methylation in the lungs. 38,39 Furthermore, evidence show that the bronchial epithelium and blood are twice as distant as the bronchial and nasal epithelium, emphasizing that DNA methylation in blood samples may not be informative enough to draw conclusions about methylation marks in the airway. 38 In the current decade, quite a few nasal methylome studies have been conducted elucidating the complex molecular patterns involved in asthma. In total, methylome studies present a great variability in demographic (age and gender distribution) and clinical characteristics (disease definition, medication, acute infection, smoking, pet exposure); however, it is noteworthy that none of the studies distinguishes subgroup of asthma and/or AR (Table 2).
Although, most studies do not include direct association to specific clinical characteristics, few of them have reduced results' variation through adjustment corrections of major components (age, gender). 40,[45][46][47] Four studies directly examined the effect of smoke on DNA methylation. Zhang et al. 46 and Qi et al., 50 did not find any association to second-hand smoking but Yang et al. 47

identified 48
DMRs that are significantly associated with environmental tobacco smoke (ETS). Furthermore, Zhu et al. 49 2019 showed that DMPs which are associated to asthma severity are also associated to second-hand smoking.
Among the first potential biomarkers in nasal epithelial cells proposed for asthma was TET1 as the hypomethylation of its promoter was associated with childhood asthma in African Americans. 45 Of note, the methylation level of this CpG site was highly correlated across nasal cells, PBMCs and saliva, making it a potential crosstissue biomarker for childhood asthma. Notwithstanding, TET1 has been found up-methylated in BECS from asthmatics compared to controls. TET1 encodes a dioxygenase that consecutively converts 5methylcytosine (5 mC) into 5-hydroxymethylcytosine (5hmC), 5formylcytosine (5 fC) and 5-carboxylcytosine (5caC), thus playing a key role in active DNA demethylation and resulting in transcriptional activation of downstream genes such as VEGFA, which is known to be associated with lung function, 52   Distinct nasal epithelial DNAm were also observed between nonsevereand severe asthma in African-American children and may be useful in predicting disease severity. Several of the annotated genes, play a critical role in asthma. [57][58][59][60][61][62] Six DMPS, which revealed to be associated with asthma severity, annotated to TMEM51, WDR25, HIPK3, and KLF11, 49 were associated with clinical features of asthma in Project VIVA, 13 supporting the involvement of these identified CpGs in regulation of asthma severity. DNAm levels of 39 DMPs significantly correlated with mRNA levels in children with RNA-seq data available. Moreover, enrichment was observed for three regulatory histone marks associated with functional gene regulatory elements around CpG sites associated with asthma severity, 49 confirming the hypothesis that histone modifications are essential for the pathogenesis and progression of asthma, which regulates gene function together with, or independent, of DNAm. 63,64 Regarding AR, data indicating its association with the methyl- annotated to ZMYND10 (a gene related to related to primary ciliary dyskinesia). 88 The methylation level of the later is negatively associated with a compared asthma-rhinitis phenotype (AsRh) suggesting that some environmental exposures could affect DNA methylation in the nasal epithelium, which may have protective effects on AsRh.
Methylation-related expression of ZMYND10 in AsRh is lower in nasal epithelial cells, or alternatively, it may be explained by a lower subset of differentiated ciliated cells in AsRh compared with healthy controls, as was recently discovered in patients with chronic rhinosinusitis through use of scRNAseq. 89 The influence of environmental exposures on the nasal epithelial epigenome was highlighted by identifying 48 DMRs in 46 unique genes that are significantly associated with environmental tobacco smoke. 47 Nevertheless, regarding AsRh, no significant associations have been shown with exposures to other potential risk factors for allergic disease, such as smoking, secondhand smoking, moulds and dampness. 50 Μicrobial species are also known to influence the epigenome, [82][83][84] for instance, respiratory virus infections affect NECs DNA methylation. Analysis of Alu methylation indicated increased global methylation occurred in NECs of asthmatics in response to virus infection. 43 The 'Inner City' genome-wide methylation analysis after rhinovirus   102 In general, allergic subjects present mainly a decreased DNAm and the CpGs are annotated to genes with biological functions relevant to allergic sensitization such as the regulation of ILs production. 103 In a large EWAS from the MEDALL consortium using four European birth cohorts and further seven validated cohorts, childhood asthma was found to be associated with a number of differentially methylated CpG positions in whole blood. 104 Nevertheless, the vast majority of the large cohorts (ewas studies) involve either whole blood or PBMCs while only a few also included blood cell populations that are sorted in limited size cohorts.
Of note, up to 40% of the differences in methylation profiles of individuals could be attributed to the cell-type heterogeneity of white blood cells. 105 The best studied type of blood cells is eosinophils; purified circulating eosinophils showed an altered DNA methylation profile, usually hypomethylation, suggesting a differential activation state which affect immune functions of this subpopulation, the interaction with other PBMCs and certain lung functions. 98,104 Genes implicated in airway remodeling, surfactant secretion and nitric oxide production in airways, as well as genes associated with cytokine production and signaling and phagocytosis in blood are characterized by decreased methylation in asthmatic subjects. 106 Four recent large epigenome wide association studies have been conducted concerning the relationship of atopy and DNA methylation. Distinct methylation signals are found between non-atopic and atopic asthma as well as between atopic and healthy subjects. In Agricultural Lung Health Study, several hundred CpG sites were differentially methylated in blood DNA from adults with non-atopic or atopic asthma compared to adults with neither asthma nor atopy while 99.5% of these CpG sites presented decreased methylation. Atopic asthma CpG sites were enriched in pathways involved in inflammatory response or characterized by chronic inflammation consistent with the inflammatory nature of atopic asthma. 107 Differential methylation has been revealed in peripheral blood associated with atopic sensitization, environmental and food allergen sensitizations (Project Viva/Generation R Study 108 and IoW study 103 during mid-childhood and adolescence, respectively; methylation sites were annotated to genes that have been implicated in asthma pathway and with biological functions relevant to allergicsensitization, mTOR signaling, inositol phosphate metabolism and the regulation of IL-5 production. 103,108 Although studies suggest that epigenetic marks in cord blood DNA may serve as early biomarkers of allergic susceptibility in childhood as some of these CpGs had nominal associations with cord blood and genes involved in airway remodeling, there are not enough data to support this suggestion. 108 The cohort of Wu et al. 109

| DNA METHYLATION COMPARISONS AMONG STUDIES AND ACROSS TISSUES
Our comparison of the aforementioned EWAS brings up some important gene loci that overlap in each tissue ( Figure 1A,B).  including whole blood and nasal samples. Considering this gene, its methylation status has also been significantly associated with total F I G U R E 1 (A) Venn diagram representing the shared differentially methylated gene loci associated with atopic asthma in nasal epithelium samples among three large EWAS: Project Viva, 13 Inner City Consortium, 47 EVA PR study. 48 B, Venn diagram representing the shared differentially methylated gene loci associated with allergic sensitization and allergic asthma in whole blood samples among four large EWAS LEGAKI ET AL.

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IgE. 113 A life course analysis of IgE hypersensitivity studying the change in methylation between cord blood and mid-childhood DNA revealed significant postnatal differentially methylated sites located within ACOT7 (four sites) and ZNF862 (three sites). 114 EPX plays a role in various immune system pathways such as the defense to microorganisms, regulation of ILs, response to oxidative stress and neutrophil degranulation. The association of EPX levels with asthma is well studied; both urinary and serum EPX levels have been linked to childhood asthma and have been suggested in the past as biomarkers. 115 Concerning IL5RA, there are plenty of data linking it with asthma in a positively correlating manner. Moreover, polymorphisms in IL5RA account as a genetic risk factors for asthma development, especially in atopic populations. 116,117 Furthermore, its function in inflammatory response, signaling transduction, like the cytokine mediated signaling pathway and MAPK cascade is known; thus, it consists a biologically plausible candidate marker for asthma development.
Unfortunately, EWAS studies that involve bronchial epithelial samples, the kind of tissue that is 'closer' to asthma, comparing methylated gene loci between healthy individuals and allergic asthmatics are scarce. In our comparisons, only one study 23 evaluating the genome-wide methylation profile of the bronchial mucosa of allergic asthmatics compared to non-allergic was included, which is apparently not sufficient.

| CONCLUSIONS
In conclusion, epigenomic and transcriptomic profiling has provided a means of exploring how gene-environment interactions contribute to the pathology of asthma and other allergic respiratory conditions.
Overall, there is significant heterogeneity among studies, in respect to sample sizes, age groups, disease phenotype, statistical methods; however, the field appears to be very conscious of limiting factors, and a steady improvement is certainly evident over time in the quality and scope of newer studies.
BECs are not easily available but more clinically relevant to airway diseases compared to other types; therefore, more EWAS studies on BECs, including comparisons between healthy individuals and atopic asthmatics, are required for more precise associations.
Distinguishing between atopic and non-atopic asthma should be taken under consideration in future studies, as epigenetic differences between the two groups can be striking. In general, accumulated evidence suggests that NECs are comparable in diagnostic F I G U R E 2 Cytoscape diagram presenting the network of common differentially methylated genes in at least three studies and the enriched pathways are implicated using reactome-pathway analysis tool Finally, it is essential to focus on more detailed investigation processes at all levels, in order to identify targeted methylation loci as potential biomarkers. The clinical characterisation of patients, the sampling procedure, the choice of technique and the analysis protocol play a role in DNA methylation results variability and repeatability. Wider studies in age groups with specific clinical characteristics and using more precise protocols will elucidate differentially methylated loci that could be used as immune age biomarkers in the future.